PB,
So now I have a question for you. In the genomeweb article on BRET used as an example in the previous post, I noticed the following:
>>That article, by Vanderbilt University biologist Yao Xu and his colleagues, said that BRET offers fewer cell-specific limitations and can more readily be used for high-throughput screening.
But the technology also has limitations, Xu warned. The proteins may interact in such a way that the luciferase and green fluorescent protein are not close enough to exchange light energy. Hence a negative result would not prove non-interaction. But this limitation is also present in other assays, he said.<<
Do you know if anybody is effectively addressing this limitation? After all, it's been almost a year.
I can get the abstract at MedLine, but PNAS Online can't seem to come up with the article:
>>A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins.
Proc Natl Acad Sci U S A 1999 Jan 5;96(1):151-6 (ISSN: 0027-8424)
Xu Y; Piston DW; Johnson CH [Find other articles with these Authors]
Department of Biology, Box 1812-B, Vanderbilt University, Nashville, TN 37235, USA.
We describe a method for assaying protein interactions that offers some attractive advantages over previous assays. This method, called bioluminescence resonance energy transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green fluorescent protein mutant fused to another protein of interest. Interactions between the two fusion proteins can bring the luciferase and green fluorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission. By using proteins encoded by circadian (daily) clock genes from cyanobacteria, we use the BRET technique to demonstrate that the clock protein KaiB interacts to form homodimers. BRET should be particularly useful for testing protein interactions within native cells, especially with integral membrane proteins or proteins targeted to specific organelles.<< snip
Cheers, Tuck |
